Regioselective liquid phase pegylation

ABSTRACT

A method is described for a method for the regioselective liquid-phase pegylation of GRF, which increases the yield of the GRF-PEG conjugate having 1 PEG molecule covalently bound to the e-amino group of Lys 12 . This method is characterized in that the reaction is carried out in a structuring solvent, such as trifluorethanol.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for the regioselectiveliquid-phase pegylation of GRF, which increases the yield of the GRF-PEGconjugate having 1 PEG molecule covalently bound to the ε-amino group ofLys¹². This method is characterized in that the reaction is carried outin a structuring solvent, as for example trifluorethanol.

BACKGROUND OF THE INVENTION

[0002] In the early 1980's several groups isolated and characterizedgrowth hormone releasing factor (GRF).

[0003] GRF (also called Somatorelin) is a peptide secreted by thehypothalamus, which acts on its receptor and can promote the release ofgrowth hormone (GH) from the anterior pituitary. It exists as 44-, 40-,or 37-amino acid peptide; the 44-amino acid form may be convertedphysiologically into shorter forms. All three forms are reported to beactive, the activity residing mainly in the first 29 amino acidresidues. A synthetic peptide corresponding to the 1-29 amino acidsequence of human GRF [GRF(1-29)], also called Sermorelin, has beenprepared by recombinant DNA technology as described in European PatentEP 105 759.

[0004] Sermorelin has been used in the form of acetate for the diagnosisand treatment of growth hormone deficiency.

[0005] GRF has indeed a therapeutic value for the treatment of certaingrowth hormone related disorders. The use of GRF to stimulate therelease of GH is a physiological method in promoting long bone growth orprotein anabolism.

[0006] One problem associated with the use of GRF relates to its shortbiological half-life (about 12 to 30 minutes). The GRF(1-29)-NH₂ issubject to enzymatic degradation and is rapidly degraded in the plasmavia dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala² andAsp³.

[0007] It is therefore advantageous to develop biologically stable, longacting GRF analogues using specific chemical modification of GRF, inorder to prevent or slow down enzymatic degradation.

[0008] Polyethylene glycol (PEG) is a hydrophilic, biocompatible andnon-toxic polymer of general formula H(OCH₂CH₂)_(n)OH, wherein n≧4. Itsmolecular weight could vary from 200 to 20,000 Dalton.

[0009] It has been demonstrated that the chemical conjugation of PEG inits mono-methoxylated form to proteins and/or peptides significantlyincreases their duration of biological action. Like carbohydratemoieties in a glycoprotein, PEG provides a protective coating andincreases the size of the molecule, thus reducing its metabolicdegradation and its renal clearance rate.

[0010] PEG conjugation is an already established methodology for peptideand protein deivery pioneered by the fundamental studies of Davis andAbuchowski (Abuchowski et al., 1977a and 1977b). PEG conjugation topeptides or proteins generally resulted in non-specific chemicalattachment of PEG to more than one amino acid residue. One of the keyissues with this technology is therefore finding appropriate chemicalmethods to covalently conjugate PEG molecule(s) to specific amino acidresidues.

[0011] Various PEG-protein conjugates were found to be protected fromproteolysis and/or to have a reduced immunogenicity (Monfardini et al.,1995; and Yamsuki et al., 1988).

[0012] One approach was recently proposed for the site-specificconjugation of PEG to low molecular weight peptides, such as GRF, whichwas prepared by solid-phase peptide synthesis. In these conjugates apegylated amino acid, prepared in advance, was introduced into thepeptide sequence during the solid-phase synthesis. This procedure,however, dramatically complicates product purification that is known tobe the critical step in solid phase synthesis. The presence of PEG, forits high molecular weight and its polydispersivity, is likely to yieldfinal products with unacceptable impurities and/or products with missingamino acids, the latter being considered to occur commonly in theMerrifield procedure.

[0013] Mono-pegylation, meaning that only one PEG molecule is attached,using solid-phase synthesis to specific amino acid residues of[Ala¹⁵]-GRF(1-29)-NH₂ has been recently reported in the literature(Felix et al., 1995). This study shows that [Ala¹⁵]-GRF(1-29)-NH₂pegylated at residues 21 or 25 retains the fill in-vitro potency of theparent [Ala¹⁵]-GRF(1-29)-NH₂. There is however no in-vivo data to showwhether these pegylated conjugates exhibit a longer duration of actionwith respect to the non-pegylated counterpart.

[0014] More recently, it has been demonstrated (Campbell et al., 1997)that the attachment of PEG with different molecular weights to theC-terminus of several analogs of GRF, again using solid-phase synthesis,had enhanced duration of action in both pig and mouse models as comparedto the non-pegylated counterpart.

[0015] EP 400 472 and EP 473 084 disclose PEG derivatives obtained byusing specific activated PEG derivatives. In the Examples also GRF ismade to react with such activated PEG derivatives, however the specificGRF-PEG which are obtained according to such patent applications are notexactly identified nor in any way characterized. There is no mention ofa specific PEG attachment site.

[0016] WO 99/27897 discloses a method for the site-specific preparationof GRF-PEG conjugates containing one or more than one PEG units (perGRF) covalently bound to Lys¹² and/or Lys²¹ and/or N^(α), characterizedin that the conjugation reaction between the GRF peptide and activatedPEG is carried out in solution and the desired GRF-PEG conjugate ispurified by chromatographic methods. According to such patentapplication the solvent was selected from the group consisting of ahighly concentrated nicotinamide aqueous solution, a buffered aqueoussolution of a defolding agent (such as urea) or a polar organic solventselected among dimethyl sulfoxide, dimethyl formamide/buffer oracetonitrile/buffer. Dimethyl sulfoxide (DMSO) was mainly used in theExamples.

DESCRIPTION OF THE INVENTION

[0017] We have now found an improvement in the method already describedin WO 99/27897, which consists in the use of a specific class ofsolvents, which will be herein defined as “structuring solvents”. Suchan improvement allows to orientate almost completely the mono-pegylationreaction towards the ε-amino group of Lys¹².

[0018] As a matter of fact, also selecting the correct molar ratioPEG/GRF, according to the present invention it is possible to obtain upto 85% of the GRF-PEG conjugate having 1 PEG molecule covalently boundto the ε-amino group of Lys¹² with respect to the total amount ofmono-pegylated species. Other mono-pegylated species could have been theGRF-PEG conjugate having 1 PEG molecule covalently bound to the s-aminogroup of Lys²¹ the GRF-PEG conjugate conjugate having 1 PEG moleculecovalently bound to the primary amino group in N^(α).

[0019] Therefore, the main embodiment of the present invention is methodfor the liquid-phase pegylation of GRF, which allows to obtainregioselectively GRF-PEG conjugate having 1 PEG molecule covalentlybound to the s-amino group Lys¹², characterized in that the reaction iscarried out in a structuring solvent.

[0020] This and other advantages of the present process over thosealready described, in particular in WO 99/27897 can be listed asfollows:

[0021] a single monopegylated form of GRF, in large excess with respectto the other monopegylated form(s), can be reproducibly obtained whenadequate reaction conditions are applied. Selectivity of theliquid-phase PEGylation of GRF procedure has been notably improved;

[0022] a regioselective acylation reaction has been obtained without theuse of protecting/deprotecting groups;

[0023] higher reaction yields are consistently observed when an excessof activated PEG is reacted with GRF. Even a three fold excess of PEG(1PEG chain per primary amine group available) does not lead to theformation of more than 10% of the poorly active in vitro dipegylated GRFform. The major species remains the monopegylated form of GRF.

[0024] According to this invention the term “GRF”, unless otherwisespecified, is intended to cover any human GRF peptides, with particularreference to the 1-44, 140, 1-29 peptides and the corresponding amidesthereof (containing an amide group at the N-terminus or C-terminus). Thepreferred GRF peptide is GRF(1-29)-NH₂.

[0025] “Structuring solvents” or “structure-promoting solvents”throughout the present invention is intended to refer to those solventsor co-solvents who are able to confer stability to the predominantconformation of a peptide or protein. Generally they are organic polarsolvents, such as alcohols, and are known to be used as solvents orco-solvents of peptides or proteins for NMR (Nuclear Magnetic Resonance)or CD (Circular Dichroism) studies.

[0026] Among the alcohols trifluoethanol (TFE) is preferred according tothe invention.

[0027] “N^(α)” throughout the present invention means the amino group atthe N-terminal position of the peptide (Tyr).

[0028] The “activated PEG” (or “pegylating agent”) is any PEGderivative, which can be used as protein modifier, because it contains afunctional group capable of reacting with some functional group in theprotein/peptide to produce the PEG-protein/peptide conjugates. A reviewof PEG derivatives useful as protein modifiers can be found in Harris(1985). The activated PEG can be an alkylating reagent, such as PEGaldehyde, PEG epoxide or PEG tresylate, or it can be an acylatingreagent, such as PEG ester.

[0029] The activated PEG is preferably used in its mono-methoxylatedform. It has preferably a molecular weight between 2,000 and 20,000.Mono-methoxylated PEG_(5,000) is particularly preferred for thepreparation of the activated PEG according to the present invention.

[0030] If activated PEG is an acylating agent, it preferably containseither a norleucine or ornithine residue bound to the PEG moiety via acarbamate linkage. These residues allow a precise determination of thelinked PEG units per mole of peptide (see for example Sartore et al.,1991). Therefore, more in particular, the preferred activated PEG ismono-methoxylated PEG_(5,000) linked by means of a carbamate bond to thealpha amino group of norleucine, that is activated at the carboxy groupas succinimidyl ester.

[0031] Another preferred activated PEG can be methoxy-PEG-succinmidylpropionate (“PEG-SPA”).

[0032] Branched PEGs are also in common use. The branched PEGs can berepresented as R(-PEG-OH)_(m) in which R represents a central coremoiety such as pentaerythritol or glycerol, and m represents the numberof branching arms. The number of branching arms (m) can range from threeto a hundred or more. The hydroxyl groups are subject to chemicalmodification.

[0033] Another branched form, such as that described in PCT patentapplication WO 96/21469, has a single terminus that is subject tochemical modification. This type of PEG can be represented as(CH₃O-PEG-)_(p)R-X, whereby p equals 2 or 3, R represents a central coresuch as lysine or glycerol, and X represents a functional group such ascarboxyl that is subject to chemical activation. Yet another branchedform, the “pendant PEG”, has reactive groups, such as carboxyl, alongthe PEG backbone rather than at the end of PEG chains.

[0034] All these branched PEGs can be “activated” as indicated above.

[0035] “Pegylation” is the reaction by which a PEG-protein/peptideconjugate is obtained starting from the activated PEG and thecorresponding protein/peptide.

[0036] The molar ratio PEG:GRF can be 1:1, 2:1 or 3:1, but 3:1 ispreferred according to the present invention.

[0037] The temperature is kept around the room temperature.

[0038] Even in this case, as for the process described in WO 99/27897,the pegylation of the present invention increases the resistance to theproteolytic degradation, does not affect, or only slightly decreases,the biological activity, depending upon the extent of pegylation andallows to obtain products (the conjugates), which are more soluble inaqueous buffered solutions.

[0039] The present invention has been described with reference to thespecific embodiments, but the content of the description comprises allmodifications and substitutions, which can be brought by a personskilled in the art without extending beyond the meaning and purpose ofthe claims.

[0040] The invention will now be described by means of the followingExamples, which should not be construed as in any way limiting thepresent invention. The Examples will refer to the Figures specified herebelow.

DESCRIPTION OF THE FIGURES

[0041] FIG. 1: it reports the semi-developed structure for the twoactivated PEGs, which have been used in the Examples.

EXAMPLES

[0042] Abbreviations

[0043] ACN Acetonitrile

[0044] DMSO Dimethylsulfoxide

[0045] GRF Growth hormone releasing factor

[0046] HPLC High Performance Liquid Chromatography

[0047] IEX Ion-exchange

[0048] mPEG-Nle-Osu Monomethoxypoly(ethylene glycol)-norleucinesuccinimidyl ester

[0049] RP Reverse phase

[0050] TEA Triethylamine

[0051] TFA Trifluoroacetic acid

[0052] TFE Trifluoroethanol (2,2,2-trifluoroethanol)

[0053] Preliminary NMR Studies on GRF

[0054] GRF is a member of a family of peptides which are known to becharacterised by a predominant α-helical secondary structure, especiallyin presence of solvents like methanol and trifluoroethanol in aqueoussolutions (see Clore et al., 1986, and Theriault et al., 1988).

[0055] Further NMR studies were more recently done at LIMA (BioindustryPark, Ivrea, Turin, Italy). One of the main aim was to better elucidatethe influence of solvents on the GRF structure, and to assess andeventual solvent-dependent conformational changes of lysine 12 andlysine 21 side-chains, changes which could modify the chemicalreactivity of the amine groups.

[0056] These studies showed that GRF structure in DMSO was poorlystructured and highly fluxional.

[0057] On the other hand, in the methanol/water 75% mixture, the globalfolding of GRF consisted of two-helix domains joined by a well-definedkink, which makes approximately a 90° angle between the axes of the-helices.

[0058] In neat trifluoroethanol, NMR studies showed a secondarystructure of GRF basically α-helical at all temperature considered wherethe 2 α-helical tracts, are joined by a central flexible segment.

[0059] A major difference with the structure in MeOH/H₂O 75% mixture, isthat this segment, joining the two α-helixes, exists in multipleconformations and is highly dependent upon the temperature.

[0060] Further studies made at different temperatures with TFE showedthat the second -helix tract (fragment 18-28) is more stable than thefirst one (fragment 4-14).

[0061] The studies showed that TFE further enhances the α-helicesstability already observed in methanol/water 75/25 mixture. Thisstabilisation appears to induce a conformational disorder in the part ofthe molecule (centred at fragment 16-19) joining them.

[0062] This structuring effect of TFE appears as a key element to thepossible orientation of the PEGylation reaction.

[0063] This structuring effect of TFE clearly appears as a key elementto the possible orientation of the PEGylation reaction. Materials andreagents Description Lot n° GRF₁₋₂₉ (Bachem) FGRF1299603Methoxy-PEG-Nle-Osu (Shearwater Polymers) PT-028-12Methoxy-PEG-succinimidylpropionate (SPA), 85969 (Fluka) Trifluoroethanol(TFE), (Aldrich), spectrophotometric grade

[0064] General Method of Synthesis

[0065] To GRF₁₋₂₉ bulk powder is added at once to trifluoroethanol.Dissolution rapidly occurs at room temperature under gentle agitation upto [GRF]=20 mg/ml in trifluoroethanol (TFE).

[0066] The chosen activated PEG reagent is then added under stirring atonce as a dry powder, or in TFE solution, to reach final PEG:GRF molarratios of 1:1, 2:1, or 3:1 (3:1 ratio is the preferred one).

[0067] The reaction mixture is then stirred at room temperature for aminimum of 6 hours. Trifluoroethanol is then evaporated under vacuum(30° C. water bath, 60 mTorr vacuum) and the reaction mixture stored at−80° C. before purification.

[0068] Results

[0069] The quenched reaction mixture of GRF, monopegylated, anddipegylated species, is analyzed by reverse-phase HPLC and therespective yield of each species determined in percentage of area. Therespective ratio of the two monopegylated species at Lysine 12 andLysine 21 is determined after purification of the crude material and inprocess analysis by reverse-phase HPLC.

[0070] Table 1 shows the results obtained at different molar ratio ofPEG and GRF in DMSO and TFE. TABLE 1 PEGylation of GRF reaction yieldsand ratio. in DMSO in TFE PEG:GRF 1:1 1.5:1 2:1 1:1 2:1 3:1 Molar ratioReaction time (h)  3 22  3  3  3 22 22  6 GRF unreacted 32 33 19  4 7058 35 26 (%*) GRF-1PEG (%*) 48 48 51 20 25 37 53 65 GRF-2PEG (%*) 20 1930 74  5  5 12  9 GRF-1PEG-Lys¹² 50 47 48 96 96 96 96 (%^(a))

[0071] Comments to the Results of Table 1

[0072] A maximum yield of 51% of GRF-1PEG-5K has been obtained inpresence of DMSO, yield which dramatically decrease (from 51 to 20%) ina two-fold excess of PEG. On the contrary and quite unexpectedly, atwo-fold excess of PEG versus GRF in presence of ThE increases the yieldof GRF-1PEG. The same observation can be made with a three-fold excessof PEG.

[0073] This example constitutes one of the main differences between theprocesses in DMSO and TFE. The reaction in TFE proceeds in aregioselective manner towards the quasi-exclusive formation of thepegylated GRF in position 12.

[0074] Table 2 indicates the ratio in % area obtained in RP-HPLC of thevarious species of the reaction mixture in two solvents,dimethylsulfoxide and trifluoroethanol, at various ratios, and with twodifferent activated PEG with the same reactive succinimidyl ester (seeFIG. 1). TABLE 2 Comparison yields and ratio of the various speciesobtained during PEGylation of GRF in DMSO and TFE solution. in DMSO InTFE PEG-Nle PEG-SPA PEG-Nle PEG-SPA PEG-SPA PEG-Nle PEG-SPA PEG:GRF 1:11:1 1:1 1.1 2:1 3:1 4:1 Molar ratio Reaction time (h) 3 3 24 24 24 24 24GRF unreacted 32 31 53.9 51 34.6 26.2 5.5 (%*) GRF-1PEG (%*) 48 48.943.6 45 57 65 64 GRF-2PEG (%*) 18 16 2.5 3.3 8.6 8.6 30.1 GRF-1PEG-Lys¹²45 45 75 79 80 82.7 85 (%^(b))

[0075] Comments to the Results of Table 2

[0076] Comparable results are obtained with either activated PEG. Thereaction in absence of TEA, proceed with a different selectivity in TFE.In presence of a three to 4-fold excess of PEG, 50% of GRF-1PEG-Lys¹²species is formed, twice as much as the maximum amount obtained in DMSO.The results obtained with a PEG:GRF ratio are the best results obtainedunder the conditions tested.

CONCLUSIONS

[0077] PEGylation of GRF process in TFE lead to the formation of a majorspecies, the GRF-1PEG-Lys¹². The use of TFE as solvent reaction wasshowed to increase the selectivity of the pegylation. TEA asnucleophilic enhancer orientates the positioning of the PEG chains ontothe GRF peptide.

[0078] Supported by the structuring effect of TFE on GRF₁₋₂₉ amide (NMRdata), possible advantages of this process over the process in DMSO arenumerous and can be summarized as follow:

[0079] a regioselective acylation reaction has been obtained without theuse of protecting/deprotecting groups;

[0080] higher yields of monopegylated GRF were experimentally obtainedin presence of TFE, with respect to the reaction in DMSO;

[0081] a large scale production of the monopegylated form of GRF willbenefit of the advantages described within this protocol.

REFERENCES

[0082] Abuchowski A. et al., J. Biol. Chem., 252, 3571-3581, 1977a;

[0083] Abuchowsli A. et al., J. Biol. Chem., 252, 3582-3586, 1977b;

[0084] Campbell R. et al. J. Peptide Res., 49, 527-537, 1997;

[0085] Clore G. M. et al., J. Mol. Biol., 191, 553-561 (1986).

[0086] Felix A. M. et al., Int. J. Peptide Protein Res., 46, 253-264,1995;

[0087] Harris J. M., Rev. Macromol. Chem. Phys., C25, pp. 325-76, 1985;

[0088] Monfardini et al., Biocon. Chem., 6, 62-69, 1995;

[0089] Sartore L. et al., Appl. Biochem. Biotechnzol., 27, 45, 1991;

[0090] Sartore L., et al., Applied Biochem. Biotechnol., 31, 213-22,1991;

[0091] Theriault Y. et al., Biopolymers, 27, 1897-1904, 1988;

[0092] Yamsuki et al., Agric. Biol. Chem., 52, 2185-2196, 1988;

[0093] Zalipsky S. et al., Advanced Drug Delivery Reviews, 16, 157-182,1995.

1 1 1 29 PRT Artificial Synthetic 1 Tyr Ala Asp Ala Ile Phe Thr Asn SerTyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu GlnAsp Ile Met Ser Arg 20 25

1. A method for the liquid-phase regioselective synthesis of the GRF-PEGconjugate having one PEG unit covalently bound to the ε amino group ofLys¹², characterized in that the conjugation reaction between the GRFpeptide and the activated PEG is carried out in a structuring solvent.2. The method of claim 1, wherein the GRF peptide is h-GRF(1-29)-NH₂. 3.The method according to anyone of the preceding claims, wherein theactivated PEG is an alkylating or acylating PEG in its mono-methoxylatedform.
 4. The method according to any of the preceding claims, in whichthe structuring solvent is trifluoroethanol.
 5. The method according toany of the preceding claims, in which the conjugation reaction iscarried out at room temperature.